Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expression

Article abstract of DOI:10.1016/j.bmc.2012.11.013

Anti-mitotic anti-cancer drugs offer a potential platform for developing new radiotracers for imaging proliferation markers associated with the mitosis-phase of the cell-cycle. One interesting target is kinesin spindle protein (KSP) - an ATP-dependent motor protein that plays a vital role in bipolar spindle formation. In this work we synthesised a range of new fluorinated-quinazolinone compounds based on the structure of the clinical candidate KSP inhibitor, ispinesib, and investigated their properties in vitro as potential anti-mitotic agents targeting KSP expression. Anti-proliferation (MTT and BrdU) assays combined with additional studies including fluorescence-assisted cell sorting (FACS) analysis of cell-cycle arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer cell lines. Additional studies using confocal fluorescence microscopy showed that these compounds induce M-phase arrest via monoaster spindle formation. Structural studies revealed that compound 20-(R) is the most potent fluorinated-quinazolinone inhibitor of KSP and represents a suitable lead candidate for further studies on designing ¹⁸F-radiolabelled agents for positron-emission tomography (PET).

Full text of DOI:10.1016/j.bmc.2012.11.013

Bioorganic & Medicinal Chemistry 21 (2013) 496–507
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorinated quinazolinones as potential radiotracers for imaging kinesin
spindle protein expression
Jason P. Holland a,b, , Michael W. Jones c, Susan Cohrs d, Roger Schibli b,d, Eliane Fischer d,
⇑
⇑
a Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Department of Radiology, Harvard Medical School, 55 Fruit St., White 427, Boston, MA
02114, United States
b Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, CH-8093, Zurich, Switzerland
c Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, United Kingdom
d Center for Radiopharmaceutical Sciences, Paul Scherrer Institute, Villigen, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-mitotic anti-cancer drugs offer a potential platform for developing new radiotracers for imaging pro-
liferation markers associated with the mitosis-phase of the cell-cycle. One interesting target is kinesin
spindle protein (KSP)—an ATP-dependent motor protein that plays a vital role in bipolar spindle forma-
tion. In this work we synthesised a range of new fluorinated-quinazolinone compounds based on the
structure of the clinical candidate KSP inhibitor, ispinesib, and investigated their properties in vitro as
potential anti-mitotic agents targeting KSP expression. Anti-proliferation (MTT and BrdU) assays com-
bined with additional studies including fluorescence-assisted cell sorting (FACS) analysis of cell-cycle
arrest confirmed the mechanism and potency of these biphenyl compounds in a range of human cancer
cell lines. Additional studies using confocal fluorescence microscopy showed that these compounds
induce M-phase arrest via monoaster spindle formation. Structural studies revealed that compound
20-(R) is the most potent fluorinated-quinazolinone inhibitor of KSP and represents a suitable lead can-
didate for further studies on designing 18F-radiolabelled agents for positron-emission tomography (PET).
Ó 2012 Elsevier Ltd. All rights reserved.
Received 3 September 2012
Revised 24 October 2012
Accepted 6 November 2012
Available online 24 November 2012
Keywords:
Quinazolinone
Kinesin spindle protein
Mitosis
Fluorine
Allosteric inhibitors
1. Introduction
and type of targets amenable for designing new anti-mitotic agents
has grown exponentially.2
Disruption of the normal processes that lead to cellular division
during mitosis and subsequent induction of cell-cycle arrest and/or
apoptosis is a key strategy in the design of modern chemotherapeu-
tic agents for use in treating cancer. Indeed, the efficacy of many
anti-cancer drugs used in the clinic relies on passive targeting of
the hyper-proliferation status (increased rate of cellular division)
of tumour versus normal tissues.1
Early development of anti-mitotic drugs focused on designing
agents that bind to microtubules (MTs) and inhibit the normal
function of the bipolar spindle. For example, common drugs which
interfere with the dynamic instability of microtubules by forming
either stabilising or destabilising interactions include taxanes
(paclitaxel), Vinca alkaloids and epothilones. However, in the clinic
use of these MT-targeted agents is often associated with severe
side-effects such as peripheral neuropathy, which limits the
maximum administered dose and duration of treatment.1 More re-
cently, as our knowledge of the cell cycle has evolved, the number
Over the last decade, kinesin motor proteins, such as kinesin
spindle protein (KSP; kinesin-5; Eg5; KIF11) have emerged as
potential targets for anti-mitotic drug development. Kinesins are
a superfamily of motor proteins that typically use the energy
derived from ATP-hydrolysis to generate force and/or transport
cargo around the cell using the MT network.3 KSP is a microtu-
bule-associated homodimeric motor protein that functions during
mitosis and is required for centrosome separation, bipolar spindle
formation, proper segregation of sister chromatids and regulation
of the rate of mitosis.4 Modulation of the activity of KSP by
immunodepletion of KSP protein, knockdown of KSP protein
synthesis by using siRNA, or specific KSP inhibition using small-
molecule agents like monastrol and ispinesib is known induce
mitosis-phase (M-phase) arrest followed by apoptosis in either
the M-phase (via mitotic catastrophe) or G1-phase of the
cell-cycle.5–7
In their landmark paper, Mayer et al.8 demonstrated that inhibi-
tion of KSP using the small-molecule inhibitor monastrol induced a
unique cellular phenotype in which the bipolar spindle formation
fails due to improper separation of the centrosomes resulting in
a characteristic monopolar (monoaster) spindle.8,9 Subsequent
structural and mechanistic studies revealed that monastrol inhibits
⇑
Corresponding authors. Tel.: +1 617 726 6107; fax: +1 617 726 6165 (J.P.H.);
tel.: +41 56 310 2857; fax: +41 56 310 2849 (E.F.).
E-mail addresses: holland.jason@mgh.harvard.edu (J.P. Holland), eliane.fischer@
psi.ch (E. Fischer).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.013

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
497
KSP by binding an allosteric site ꢀ12 angstroms away from to the
ATP-binding pocket.10,11 Further chemical screening for novel
pharmacophores specific for KSP inhibition led to the identification
of numerous classes of structurally diverse compounds including
the potent quinazolinone-based compound, ispinesib which has
been evaluated in numerous Phase
I and II clinical trials
(Fig. 1).12–15
In our efforts toward developing radiolabelled agents for use in
positron-emission tomography (PET) imaging of cancer, we rea-
soned that a radiotracer targeting KSP has the potential to inform
medicinal chemists about drug transport, targeting and delivery,
and could also provide a novel strategy for imaging tumour prolif-
eration status by targeting cells in the M-phase. In this work we
present details of the synthesis and preliminary in vitro evaluation
of fluorinated quinazolinone derivatives based on the structure of
ispinesib which have potential to be radiolabelled with fluorine-
18 for developing new PET radiotracers.
Figure 2. Diagram showing the potential sites for synthesising fluorinated quinaz-
olinone derivatives of ispinesib which are potentially amenable to common 18F-
radiolabelling strategies. Substitution sites (i–iv) are discussed in the main text.
2. Results and discussion
2.1. Compound design
substitution of a leaving group with 18F anions.16 Examination of
the crystal structure of ispinesib bound in the allosteric pocket of
KSP also revealed that functionalisation of the benzyl group would
likely induce a loss of binding affinity due to steric interactions
with the protein.14 Based on crystallographic data, and structure-
activity relations, functionalisation of the para-methylbenzamide
group (position iii) or the free primary amine which is solvent ex-
posed (position iv) is also feasible. Since 18F-radiolabelling of the
primary amine would require substantial structural modification
involving the use of a radiolabelled prosthetic group such as amide
coupling to [18F]-SFB,17 we elected to modify the para-methylben-
zamide group in the synthesis of our fluorinated-derivatives.
Functionalisation of the para-methylbenzamide group has four
distinct advantages. First, the aromatic ring in position iii is deac-
tivated due to the electron withdrawing amide substituent which
would facilitate SNAr reactions for nucleophilic incorporation of
18F. Second, the F and NO2 derivatives can be introduced at a
late-stage in the synthesis. Third, introducing fluorine in the
ortho-, meta- and para-positions requires changing only one syn-
thetic step and facilitates the study of structure-activity relation-
ships. Fourth, it has been demonstrated that substitution of the
methyl group for Br in CK-0106023 (Figure 1) does not interfere
with the specificity or affinity of this compound for KSP.9
In designing a potential 18F-radiopharmaceutical based the
quinazolinone pharmacophore of ispinesib, we considered
four possible locations for introducing the fluorine substituent
(Fig. 2): (i) substitution of the Cl group on the quinazolinone ring,
(ii) functionalisation of the benzyl group, (iii) replacement of the
methyl substituent of the para-methylbenzamide group, and (iv)
derivatisation of the free primary amino group. As 18F is most
commonly available as a nucleophilic source and the majority of
18F-radiolabelling protocols employ nucleophilic substitution reac-
tions,16 it was important for our synthetic route to be amenable to
incorporation of a suitable leaving group (in this case, NO2).
Replacement of the Cl group of the quinazolinone ring with F or
NO2 (for synthesis of a precursor molecule suitable for radiolabel-
ling) is feasible (Figure 2, position i). However, synthesis was
prohibited by solubility issues as well as limited availability of
sufficient starting materials (data not shown). In addition, intro-
duction of a functional group modification at the first synthetic
step is less desirable than at a late-stage. Functionalisation of the
benzyl moiety (position ii) is feasible. Yet when considering
subsequent 18F-radiolabelling via an SNAr reaction, the electron
rich, non-activated aromatic ring is likely to hinder efficient
Figure 1. Structures of selected allosteric KSP inhibitors.

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J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
2.2. Chemical synthesis
Section 4 (vide infra). Where appropriate, the structural identity
and purity of all novel compounds has been characterised by using
a range of experimental techniques including thin-layer chroma-
tography (TLC), 1H, 13C{1H} and 19F NMR spectroscopy, polarimetry
and high resolution electrospray ionisation mass spectrometry.
The optimised synthesis of enantiomerically pure compounds
19–22 was accomplished in 11 steps in accordance with Scheme 1.
Full synthetic details and characterisation data are given in the
Scheme 1. Reaction scheme for the synthesis of quinazolinone compounds 14–22. Reagents and conditions: (i) Compounds 1 and 2, THF, stir, N2(g), rt, 2 h, (ii) Crude
intermediate 3, acetic anhydride 4, stir, N2(g), 100 °C, 5 h, (iii) Compounds 5 and 6, toluene, Dean–Stark reflux, N2(g), 3 h, then add ethylene glycol and continue Dean–Stark
reflux, 5 h, (iv) Compound 7, NaOAc, glacial acetic acid, Br2/acetic acid (dropwise) over 30 min, then stir, 45 °C, 4 h, (v) Compound 8, NaN3, anhydrous DMF, stir, 45 °C, 5 h, (vi)
Compound 9, EtOAc/MeOH (30 mL/100 mL), NH4Cl(s), activated Zn(s) powder, stir, 60 °C, 30 min, (vii) Chiral resolution: for 10-(R): Racemate 10, (R,R)-(+)-2,3-dibenzyl-
D-
tartaric acid, propan-2-ol, stir, 85 °C, then cool to rt, 18 h. For 10-(S) the same procedure was employed using (S,S)-(ꢁ)-2,3-dibenzyl-
L
-tartaric acid, (viii) Swern oxidation,
oxalyl chloride, anhydrous DCM, DMSO, stir, Ar(g), ꢁ78 °C, then alcohol 11 (dropwise), ꢁ78 °C, followed by NEt3, ꢁ78 °C to rt over 1 h, (ix) Compounds 10 and 12,
NaBH(OAc)3, 1,2-dichloroethane, stir, Ar(g), rt, 6 h, (x) BOC-protected derivatives 14–18: Compound 13 and substituted benzoyl chlorides (R1 = p-CH3, p-F, m-F, o-F, p-NO2),
DIPEA, anhydrous DCM, stir, rt, 24 h, (xi) BOC-deprotection: Compound 14–18, trifluoroacetic acid, DCM, stir, N2(g), rt, 2 h.

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
499
Figure 3. Representative images of the single crystal X-ray diffraction structures of (A) intermediate 3, (B) compound 7 and (C) compound 9. Thermal ellipsoids are shown at
50% probability. Hydrogen atoms have been omitted for clarity.
Compound 5 was isolated in 88% yield from a two-step reaction
via intermediate 3 starting from 4-chloroanthranilic acid 1 and iso-
valeryl chloride 2. Although isolation of intermediate 3 is possible
and single crystals of 3 were obtained (Fig. 3A), reaction of the
crude material with acetic anhydride proceeded in high yield.
Reaction of compound 5 with benzyl amine under Dean–Stark
Following the Swern oxidation of compound 11, and the reduc-
tive amination of compound 10 in the presence of compound 12
and NaBH(OAc)3 furnished compound 13 in ꢀ75% yield. Here, com-
pound 13 was used as a common intermediate for all derivatives,
thus minimising the number of synthetic steps required after
introducing the different substituents. Notably, introduction of
the para-nitro substituent in the BOC-protected compound 18
occurs as the final step for the synthesis of the 18F-radiolabelling
precursor. Finally, deprotection using trifluoroacetic acid yielded
compounds 19–22 in overall yields ranging from 21% to 38% over
a total of 10 or 11 steps. Compounds 19–22 were evaluated for
their potential to inhibit KSP by using a range of protein and
cellular-based assays in vitro.
reflux conditions afforded compound 7 in 84% yield. Then
a-bro-
mination followed by nucleophilic substitution with sodium azide
gave compounds 8 and 9, respectively. Single-crystal X-ray struc-
tures of key cyclized quinazolinone compound 7 and the racemic
azide compound 9 are shown in Figure 3B and C, respectively.
The azide group of compound 9 was subsequently reduced to a
primary amine by using Zn/NH4Cl to give racemate 10 in near
quantitative yield over 3 steps from compound 7.
Chiral resolution of 10 using enantiomers of 2,3-dibenzyl-
D
-tar-
2.3. Kinesin motor protein inhibition assays
taric acids, afforded enantiomerically pure compounds 10-(R) and
10-(S). The absolute configuration of the chiral centre, and enantio-
meric purity of compound 10-(R) and 10-(S) was confirmed after
synthesising the Mosher’s amide derivatives 23-(R,R) and 23-
(R,S) (Scheme 2). 1H NMR analysis was conducted in accordance
with the methods described by Hoye et al.18 Both compounds
23-(R,R) and 23-(R,S) were obtained in greater than 99% de, con-
firming the enantiomeric purity of the parent compounds 10-(R)
and 10-(S).
After successful isolation of the fluoro-quinazolinone
derivatives 20–22, we first investigated their ability to inhibit the
enzymatic ATP-hydrolysis activity of KSP, as well as their selectiv-
ity for KSP versus a panel of diverse kinesin motor proteins (Table 1
and Supplementary data Fig. S1). Kinesins investigated included
centromeric protein-E (CENP-E), mitotic centromere-associated
kinesin (MCAK), kinesin heavy chain (KHC) and kinesin protein
KIFC3. In addition, (S)-monastrol and (R)-ispinesib (19-(R)) were
Scheme 2. Reaction scheme showing the synthesis and structures of Mosher’s amides I and II derived from amide coupling of compound 10-(R) with R-(+)-MTPA-OH or S-
(ꢁ)-MTPA-OH to give compounds 23-(R,R) and 23-(R,S), respectively. 1H NMR resonance assignments are presented (letters a–g) and chemical shift data are given in the table
(inset). NB: Analysis and assignment of the absolute stereochemical configuration of compound 10-(R) was accomplished in accordance with the methods of Hoye et al.18
where R1 = isopropyl group and R2 = quinazolinone ring.

500
J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
Table 1
Percentage of kinesin motor protein ATP-hydrolysis activity relative to untreated controls/%
Compound
Relative kinesin motor protein ATP-hydrolysis activity/%
KSP
CENP-E
MCAK
97 2.1
KHC
98.5 1.0
KIFC3
(S)-Monastrol (10
lM)
10.8 1.4
8.8 1.4
99.4 1.1
2.7 1.7
96.1 2.6
10.1 3.2
95.5 3.0
7.5 2.4
95.9 3.4
97.7 2.2
98 3.7
98.3 2.6
97.3 2.8
97.9 2.5
101.3 1.9
98.7 1.9
99.4 1.4
98.9 1.8
99.4 2.0
98.8 2.1
19-(R) (1
19-(S) (1
20-(R) (1
20-(S) (1
21-( ) (1
21-(S) (1
22-(R) (1
22-(S) (1
l
l
l
l
l
l
l
l
M)
M)
M)
M)
M)
M)
M)
M)
96.7 3.2
99.2 2.3
99.2 2.4
98.9 1.8
96.4 2.5
98.6 1.2
98.7 3.1
98.2 2.1
101.5 4.3
96.8 2.3
97.1 3.3
99.2 2.0
99.1 2.1
98.9 1.8
97.2 3.2
97.9 1.9
99.1 1.6
99.8 1.6
97.4 2.0
97.4 1.9
100.5 1.7
99.1 1.7
97.9 2.0
Table 2
Data on the GI50 values for growth inhibition in four human cancer cell lines by compounds 20–22 as measured by using MTT assays
Compound
DU-145 cells
PC-3 cells
MCF-7 cells
GI50(2)/nM R2
SKOV-3 cells
GI50(2)/nM
GI50(1)/
l
Ma GI50(2)/nMb R2
GI50(1)/
lM
GI50(2)/nM R2
GI50(1)/
lM
GI50(1)/
lM
R2
20-(R)
20-( )
20-(S)
21-( )
21-(S)
22-(R)
22-( )
22-(S)
2.2 2.4
3.1 3.6
0.35 0.05
3.3 14.9
0.52 0.09
5.6 17.8
NDc
0.19 0.1
1.1 0.1
N/Ad
4.0 0.6
N/A
10.7 1.2
48.5 7.5
N/A
0.968
5.1 1.2
6.3 1.5
1.3 0.2
7.6 2.8
1.8 0.2
4.6 1.5
0.5 0.1
1.1 0.3
N/A
17.0 7.3
N/A
7.8 3.7
1.9 0.8
N/A
0.933
2.5 1.1
2.7 1.1
1.1 0.2
2.9 1.4
1.0 0.2
4.0 0.6
N/A
5.1 0.8
N/A
4.2 0.7
17.7 3.6
N/A
0.946 15.4 4.5
0.00226 0.0004 0.967
0.958
0.953
0.961
0.918
0.979
0.938
0.898
0.915
0.969
0.958
0.970
0.898
4.3 2.5
0.5 0.05
5.7 3.2
1.1 0.2
2.7 0.6
N/A
0.932
0.981
0.968
0.923
0.913
0.960
0.946
15.6 3.2
N/A
0.965 0.63 0.12
0.909
0.961
0.942
3.0 1.3
1.9 1.3
2.0 0.4
0.970 5.35 6.4
0.971 3.1 2.7
0.877 0.85 0.12
34.8 13.6
34.6 9.5
N/A
0.945 0.35 0.1
0.946 0.16 0.02
0.72 0.10
NB: Where two separate GI50 values are given, a biphasic model was employed. For (S)-enantiomers a single value derived from monophasic non-linear regression analysis is
given.
a
GI50 value for non-specific cytotoxicity in lM.
GI50 value for M-phase KSP inhibition of cell-cycle progression (cytostatis).
Not determined.
Not applicable.
b
c
d
used as positive controls, and the inactive stereoisomer (S)-ispine-
sib 19-(S) was used as a negative chemical control for KSP
inhibition.
(GI50(2)/nM) concentration ranges (Figs. 4A and 5). For analysis
of growth inhibition curves derived from cells treated with (S)-
enantiomers, a monotonic sigmoid shape was used with non-linear
regression analysis yielding one measure of growth inhibition
These assays indicated that only the (R)-enantiomers of com-
pounds 19–22 are active toward KSP inhibition in vitro. Further,
the (R)-enantiomers of compounds 19–22 were found to be selec-
tive for KSP inhibition and failed to inhibit the ATP-hydrolysis
activity of CENP-E, MCAK, KHC or KIFC3. Interestingly, structure-
activity relations suggest that the fluorine atom can be located in
the ortho-, meta- or para-position (Scheme 1) without compromis-
ing binding or selective inhibition of KSP.
(GI50(1)/
lM) in the micromolar range. At relatively high concentra-
tions (typically >0.5–10
l
M) all compounds were found to be cyto-
toxic. The mechanism of induced cell death is uncertain but is
tentatively assigned to general cytotoxic effects from treatment
with high concentrations of these agents. For the (S)-enantiomers,
treatment with drug concentrations <0.1 lM did not affect cellular
growth/proliferation with absorbance values corresponding to
those measured for control (vehicle treated) samples. However,
treatment with the (R)-enantiomers and racemates resulted in a
dramatically different profile; below the cytotoxic threshold at
concentrations in the range ꢀ1 to ꢀ10 nM, a plateau was observed.
At concentrations below the plateau (<10 nM) we observed a sec-
ond sigmoidal profile with measured absorbance increasing to that
observed in vehicle-treated control samples. Non-linear regression
analysis of this biphasic profile yielded GI50(2)/nM values in the
nanomolar range (Table 2 and Fig. 5). In the concentration range
corresponding to the plateau between the GI50(1) and GI50(2) val-
ues, (R)-enantiomers of compounds 20–22 likely inhibit cell-cycle
progression by inducing cytostasis in the M-phase from KSP inhibi-
tion. It is plausible that at sub-nM concentrations, these drugs are
not present in sufficiently high amounts to effectively inhibit all
KSP motor protein present, and therefore, cell-cycle progression
proceeds as in control samples.
2.4. Cellular growth inhibition assays
We next investigated the potency of the fluoro-compounds 20–
22 toward cellular growth inhibition in DU-145 and PC-3 prostate
cancer, MCF-7 breast cancer and SKOV-3 ovarian cancer cells
(Table 2 and Fig. 4). Previous work on KSP inhibition has focused
on the use of drugs like ispinesib to inhibit the growth of colorectal
carcinomas (using HCT116 cells) and ovarian cancer (SKOV-3
cells).9 These two cell lines were found to be particularly sensitive
toward KSP inhibition. In this work, we were also interested in
evaluating the potential scope of our novel quinazolinone com-
pounds toward growth inhibition of different cancer cell lines.
Cell proliferation data indicate that after 44 h incubation, com-
pounds 20–22 were found to inhibit growth of all four cell lines.
The (R)-enantiomers of all compounds were most active, and in
general, the para-fluoro derivative (compound 20) was more po-
tent than either the meta- or ortho-isomers (compounds 21 and
22, respectively). Analysis of dose–response curves for treatment
with the (R)-enantiomers or racemates showed a biphasic profile
with non-linear regression analysis yielding two growth inhibition
Structure–activity relationships for growth inhibition studies
showed the same trend of potency for all compounds tested in
DU-145, PC-3 and MCF-7 cells. For each of these three cell lines,
compound 20-(R) was the most potent with GI50(2) values ranging
from 0.19 nM (DU-145 cells) to 0.98 nM (MCF-7 cells). Our results
also demonstrate that of the cancer cell lines tested, SKOV-3 cells
values centred in the micromolar (GI50(1)/lM) and nanomolar

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
501
Figure 4. Representative growth inhibition curves showing the effect of enantiomerically pure and racemic mixtures on growth inhibition of MCF-7 breast cancer cells for (A)
para-fluoro compound 20 and (B) ortho-fluoro compound 22.
propidium iodide (PI). In addition, we used immuno-staining of
phospho-histone H3 (pH3), a specific marker of mitosis,19 to
deconvolute changes in the cell population numbers in the G2/M-
phase.
DNA content analysis demonstrated that (S)-enantiomers of
compounds 19 and 20 do not affect the relative distribution of cells
in the G0/G1, S and G2/M-phases of the cell cycle (Fig. 6A). Further,
treatment of cells with (S)-enantiomers at 100 nM concentration
does not induce cell death as demonstrated by the absence of cells
in the sub-G1 population. In contrast, treatment with the active
(R)-enantiomers of compounds 19 and 20 induced a dramatic shift
in the population profiles. For both compounds, the number of cells
found in the G2/M-phase increased, with a concordant decrease in
the G0/G1 phase. For instance, treatment of SKOV-3 cells with com-
pound 20-(R) induced an increase in the G2/M-phase population
from 21.8% (vehicle) to 58.5% whereas for compound 20-(S) the
population remained at 22.5% (Table 3). Notably, FACS data for
treatment with compounds 19-(R) and 20-(R) also showed an in-
crease in the number of cells associated with the sub-G1 popula-
tion which is assigned to induced apoptosis (likely resulting from
mitotic catastrophe). Similar results were found across all cell lines
studied.
An increase in the G2/M-phase population does not necessarily
confirm the mechanism of inhibition. Therefore, we conducted pH3
staining to evaluate the contribution from cells in the M-phase
(Figs. 6B and C). From the 2-dimensional scattergrams we observed
that treatment of SKOV-3 cells with compound 20-(R) resulted in
58.8% of cells associated with the M-phase. In contrast, treatment
with compound 20-(S) resulted in only 2.6% of cells in mitosis.
These FACS data demonstrate that the anti-proliferative effects of
(R)-quinazolinone-based agents are the result of induced M-phase
arrest. Further, our data are consistent with the known mechanism
of action of KSP inhibition with other inhibitors including monas-
trol, ispinesib and progenitor biphenyl compounds.8,9,20
Figure 5. Detailed comparison of the biphasic growth inhibition profile observed in
MTT and BrdU assays on treatment of SKOV-3 cells with compound 20-(R).
are by far the most sensitive toward KSP inhibition with compound
20-(R) (Fig. 5 and Table 2). MTT assays revealed that compound 20-
(R) has the same general cytotoxic value as the other compounds/
isomers tested with GI50(1) = 15.4 4.5 lM, but a significantly
lower GI50(2) value for growth arrest of only 0.00226
0.0004 nM in SKOV-3 cells. This pico-molar potency of compound
20-(R) toward induced growth arrest in SKOV-3 cells was con-
firmed by using independent BrdU growth inhibition assays which
gave GI50(1) and GI50(2) values of 61.0 27.0 lM and 0.010 0.004
nM, respectively. NB: All assays were performed in quadruplicate to
minimise contributions from irreproducible errors. Overall, these
results are consistent with previous reports which noted that
SKOV-3 cells are particularly sensitive toward KSP inhibition with
quinazolinone-based drugs.9 At present, the reasons why SKOV-3
cells display such extreme sensitivity toward KSP inhibition with
compounds 19-(R) and 20-(R) remain uncertain.
2.6. Confocal fluorescence microscopy
2.5. Cell-cycle analysis
After establishing the relative sensitivity of various human can-
cer cell lines toward treatment with compounds 20–22, we next
investigated the induced phenotypic effects by using confocal fluo-
rescence microscopy. KSP is required for bipolar spindle formation
and force generation on interpole MTs.21 Inhibition of KSP induces
M-phase arrest during prophase/prometaphase, which prevents
separation of the two centrosome to opposite poles of the cell.
Thus, KSP inhibition leads to a failure to establish a functional
bipolar spindle, and gives rise to a characteristic monoaster
phenotype.8
In order to confirm that the anti-proliferative effects observed
in the MTT and BrdU assays were the result of inhibition of cellular
proliferation in the M-phase, we investigated changes in cell-cycle
population numbers on treatment with the (R)- and (S)-enantio-
mers of the most potent new compound 20 by using
fluorescence-assisted cell sorting (FACS; Fig. 6 and Table 3). The
(R)- and (S)-enantiomers of ispinesib (compound 19) were used
as positive and negative chemical controls, respectively. Changes
in DNA content, and hence, the number of cells associated with a
given phase of the cell-cycle were measured by staining with
Human cancer cells were grown for 22 h on 8-well chambered
microscope slides before treating with compounds 20–22 (100

502
J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
Figure 6. Representative cell-cycle analysis data derived from FACS studies on treatment of SKOV-3 cells with the (R)- and (S)-enantiomers of compounds 19 and 20. (A)
Stacked plot of 1-dimensional histograms showing the change in DNA content, hence shift in the percentage of cells found in the G0/G1, S and G2/M phase of the cell-cycle
between vehicle-treated control cells and cells treated with quinazolinone-based drugs (100 nM). PI = Propidium iodide. 2-Dimensional scattergrams showing the change in
phospho-histone H3 (pH3) stained cells versus DNA content (PI staining) for (B) treatment with compound 20-(R), and (C) treatment with compound 20-(S).
Table 3
Data showing the measured percentage of cells in the G1/G0, S and G2/M phases of the cell-cycle upon treatment with either vehicle (<1% DMSO in medium) or compounds 20–22
(100 nM) at 37 °C for 24 h
Compound
DU-145 cells
S
PC-3 cells
S
MCF-7 cells
S
SKOV-3 cells
S
G0/G1
G2/M
G0/G1
G2/M
G0/G1
G2/M
G0/G1
G2/M
Vehicle
19-(R)
19-(S)
20-(R)
20-(S)
53.2
24.8
53.7
ND
52.9
4.5
31
32.6
13.8
34.4
12.0
31.0
14.2
10.1
30.0
11.1
24.2
26.3
60.9
25.0
63.6
32.9
48.2
22.1
49.7
25.1
51.3
19.6
10.6
17.6
12.0
15.2
22.4
57.2
21.2
53.2
22.8
51.1
3.8
49.8
3.5
18.4
13.6
16.6
13.8
16.2
21.8
51.3
14.5
58.5
22.5
10.8
10.3
ND
49.8
24.5
ND
a
9.5
27.7
51.9
a
ND = Not determined.
spindle with chromosomes aligned on the metaphasic plate
(Fig. 7B). In contrast, no cells in the normal stages of mitosis were
identified after treatment with compound 20-(R). Instead, we ob-
served that the majority of treated cells exhibited a monoaster
phenotype (Fig. 7A). Results were confirmed by using (R)-ispinesib
and (S)-ispinesib as positive and negative controls, respectively
(data not shown). Interestingly, after 22 h incubation virtually all
SKOV-3 cells treated with (R)-enantiomers of compounds 20–22
displayed a monoaster phenotype. These data are congruent with
the results obtained from anti-proliferative MTT and BrdU assays
as well as from the FACS studies where we observed a biphasic
growth inhibition profile and a plateau corresponding G2/M-phase
to cell-cycle arrest.
Taken together, our experimental data provide compelling evi-
dence that the anti-proliferative effects of compounds 20–22 are
consistent with the known mechanism of action of the parent drug
ispinesib. Further, we conclude that introduction of the para-fluoro
substituent in compound 20 is tolerated and does not adversely af-
fect the specificity or potency of this compound toward KSP inhibi-
tion in vitro. Collectively, the results presented here provide strong
support for the future development of an 18F-radiolabelled ana-
logue of compound 20-(R) as a potential PET radiopharmaceutical
for imaging KSP expression and monitoring tissue proliferation sta-
tus in vivo.
Figure 7. Representative confocal fluorescence microscopy images showing the
effects of treatment of SKOV-3 cells with compound 20-(R) at 100 nM after
incubation at 37 °C for 22 h. (A) Two cells displaying monoaster MT spindle
formation (red) and diffuse staining for DNA (green)—a characteristic phenotype
indicative of KSP inhibition and M-phase arrest. (B) Representative images of a
vehicle-treated SKOV-3 cell undergoing normal mitosis (anaphase). Full microscopy
details are given in the Section 4.
nM) or vehicle (<1% DMSO in medium) at 37 °C for 24 h. SKOV-3
cells were then fixed and permeabilised before staining with an
anti-a-tubulin antibody (and a secondary antibody labelled with
Alexa Fluor 568) to probe the structure of the MT-network, and
Hoechst 33342 to stain for DNA. Representative confocal fluores-
cence microscopy images showing the effect of treating SKOV-3
cells with compound 20-(R) are presented in Figure 7.
In the control (vehicle-treated) SKOV-3 cells, we observed cells
undergoing normal mitotic division with a well-defined bipolar
3. Conclusions
Here we report the synthesis and in vitro characterisation of a
range of fluorinated quinazolinone compounds based on the struc-
ture of ispinesib, a potent KSP inhibitor. Introduction of a fluorine

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
503
substituent in place of the methyl group of the para-methylbenza-
mide moiety of ispinesib was well-tolerated with compounds 20–
22 retaining specificity and potency toward KSP protein inhibition
and anti-proliferative activity in a range of human cancer cell lines.
Structure-activity studies revealed that the para-fluoro isomer was
generally more potent than the meta- or ortho-fluoro derivatives
and represents a suitable lead compound for developing a novel
18F-radiotracer for PET imaging of KSP expression and tissue prolif-
eration status. Work on the radiosynthesis of [18F]-20-(R) from the
para-nitro precursor compound 18-(R) is underway.
(p-Ph), 128.3 (Ar: –CH–CH–C(Cl)CH–), 128.9 (2C, Ar: 2 ꢂ CH3CHCH),
129.1 (Ar: –CH–CH–C(Cl)CH), 129.4 (2 ꢂ m-Ph), 133.9 (IV° C), 137.0
(IV° C), 139.7 (IV° C), 141.0 (IV° C), 147.7 (IV° C), 156.1 (IV° C), 162.3
(IV° C), 173.2 (IV° C). MS m/z (MeCN) 617 (100%) [M+H+]. HRMS-ESI
m/z (DCM/MeOH) found 617.2893, calcd for
C35H42ClN4O4
617.2889 [M+H+]. [
a
]
D
(25 °C; CHCl3; 14-(R)) = +269.7 0.7. Ana-
lytical data for the synthesis of 14-(R) and 14-(S) were identical.
4.1.2. (R)-tert-Butyl (3-(N-(1-(3-benzyl-7-chloro-4-oxo-3,4-
dihydroquinazolin-2-yl)-2-methylpropyl)-4-fluorobenzamido)
propyl)carbamate 15-(R)
Compound 13-(R) (0.150 g, 0.30 mmol) was reacted with p-flu-
orobenzoyl chloride (0.067 g, 0.42 mmol, 1.4 equiv) in accordance
with general method A to give compound 15-(R) as a white solid
(0.135 g, 0.218 mmol, 73%). 1H NMR (400 MHz, CDCl3): dH 0.31
4. Experimental
Full details are reported in the Supplementary data.
4.1. Synthesis
3
(d, 3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2), 0.74
3
(m, 1H, –CH2CH2CH2 diastereotopic), 0.89 (d, 3H, JHH = 6.7 Hz,
magnetically in equivalent CH(CH3)2), 1.11 (m, 1H, –CH2CH2CH2
diastereotopic), 1.35 (s, 9H, –OC(CH3)3), 2.53–2.73 (m, 3H, overlap-
ping resonances: 1H, CH2CH(CH3)2, and 2H, NCH2CH2CH2–), 3.25–
3.48 (m, 2H, NCH2CH2CH2–), 3.97 (br s, 1H, NHC(O)), 5.16 (d, 1H,
2JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.64 (d, 1H,
Synthetic methods and characterization data for compounds 5,
7–10, 12 and 13 are presented in the Supplementary data.
4.1.1. General method A
2
3JHH = 10.6 Hz, CHCH(CH3)2), 6.06 (d, 1H, JHH = 15.7 Hz, roofing
BOC-protect derivatives of ispinesib were synthesised by using
the same general method A given here for the synthesis of ( )-tert-
butyl (3-(N-(1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-
2-yl)-2-methylpropyl)-4-methylbenzamido)propyl)carbamate,
racemate 14.
diastereotopic PhCH2), 7.03–7.27 (m, 7H, 4 ꢂ Ar-CH, o-Ph and p-
Ph), 7.33 (m, 2H, m-Ph), 7.44 (dd, 1H, 3JHH = 8.6 Hz and w-coupling
4JHH = 2.0 Hz, Ar: –CH–CH–C(Cl)CH–), 7.71 (d, 1H, w-coupling
3
4JHH = 2.0 Hz, Ar: CH–C(Cl)–CHC(N)), 8.26 (d, 1H, JHH = 8.6 Hz, Ar:
C–CH–CH–C(Cl)). 13C{1H} (100 MHz, CDCl3): dC 18.4 (CH3), 19.2
(CH3), 28.4 (3 ꢂ CH3), 29.0 (CHCH(CH3)2: HSQC), 31.1 (–
NCH2CH2CH2–), 37.7 (–NCH2CH2CH2–), 42.2 (–NCH2CH2CH2–),
45.6 (PhCH2), 59.8 (CHCH(CH3)2), 79.5 (OC(CH3)3), 115.8 and
116.0 (2C, Ar: 2 ꢂ F-CCH), 119.4 (IV° C), 126.9 (Ar: CH–C(Cl)–
CHC(N)), 127.3 (2 ꢂ o-Ph), 127.8 (p-Ph), 128.2 (Ar: –CH–CH–
C(Cl)CH–), 128.3 (2C, Ar: 2 ꢂ F-CCHCH), 128.9 (2 ꢂ m-Ph), 129.0
(Ar: –CH–CH–C(Cl)CH), 136.8 (IV° C), 141.0 (IV° C), 147.6 (IV° C),
155.8 (IV° C), 161.9 (IV° C), 162.2 (IV° C), 164.4 (IV° C), 172.0 (IV°
C). 19F NMR (376 MHz, CDCl3; CFCl3 reference): dF ꢁ110.10 ppm.
MS m/z (MeCN) 621 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH)
To a flame dried 25 mL round bottom flask was added com-
pound 13 (0.15 g, 0.30 mmol), anhydrous dichloromethane
(2.5 mL) and diisopropylethylamine (DIPEA; 0.097 g, 0.75 mmol,
2.5 equiv) in anhydrous dichloromethane (1 mL). Then p-toluoyl
chloride (0.065 g, 0.42 mmol, 1.4 equiv) was dissolved in anhy-
drous dichloromethane (<2 mL) and added to the reaction which
was subsequently stirred at rt for 24 h. Progress of the reaction
was monitored by TLC (30% EtOAc/hexanes/ꢀ0.1% conc. NH3, UV)
with the product observed at Rf = 0.39. The reaction was quenched
by pouring onto sat. NaHCO3(aq) (30 mL), and extracted with
dichloromethane (3 ꢂ 20 mL). The organic fractions were com-
bined, washed with brine (1 ꢂ 30 mL), dried over Na2SO4(s) for
30 min, filtered and then the solvent was removed under reduced
pressure. The extraction process removed several of the major
impurities observed near the baseline by TLC analysis. The crude
mixture was purified by using silica gel chromatography (15–20%
EtOAc/hexane/ꢀ0.1% conc. NH3). Fractions containing the product
were identified by TLC, pooled and the solvent was removed under
reduced pressure. Then the sample was dried in vacuo to give com-
pound 14 as a white, microcrystalline solid (0.167 g, 0.27 mmol,
90%). 1H NMR (400 MHz, CDCl3): dH 0.36 (d, 3H, 3JHH = 6.4 Hz, mag-
netically inequivalent CH(CH3)2), 0.73–0.90 (m, 2H, –CH2CH2CH2),
found 621.2638, calcd for C34H39ClFN4O4 621.2638 [M+H+]. [
a]
D
(25 °C; CHCl3; 15-(R)) = +249.9 0.7. Analytical data for the syn-
thesis of racemate 15 and 15-(S) were identical.
4.1.3. ( )-tert-Butyl (3-(N-(1-(3-benzyl-7-chloro-4-oxo-3,4-
dihydroquinazolin-2-yl)-2-methylpropyl)-3-fluorobenzamido)
propyl)carbamate 16
Compound 13 (0.10 g, 0.20 mmol) was reacted with m-fluor-
obenzoyl chloride (0.044 g, 0.28 mmol, 1.4 equiv) in accordance
with general method A to give compound 16 (0.079 g, 0.127 mmol,
63%). 1H NMR (400 MHz, CDCl3): dH 0.37 (d, 3H, 3JHH = 6.4 Hz, mag-
netically inequivalent CH(CH3)2), 0.78 (m, 1H, –CH2CH2CH2 diaste-
3
0.88 (d, 3H, JHH = 6.6 Hz, magnetically inequivalent CH(CH3)2),
3
1.41 (s, 9H, –OC(CH3)3), 2.38 (s, 3H, PhCH3), 2.58–2.78 (m, 3H,
overlapping resonances: 1H, CH2CH(CH3)2, and 2H, NCH2CH2CH2–
), 3.33–3.51 (m, 2H, NCH2CH2CH2–), 3.88 (br s, 1H, NHC(O)), 5.23
reotopic), 0.95 (d, 3H, JHH = 6.7 Hz, magnetically inequivalent
CH(CH3)2), 1.31 (m, 1H, –CH2CH2CH2 diastereotopic), 1.41 (s, 9H,
–OC(CH3)3), 2.59–2.79 (m, 3H, overlapping resonances: 1H,
CH2CH(CH3)2, and 2H, NCH2CH2CH2–), 3.29–3.53 (m, 2H,
NCH2CH2CH2–, diastereotopic), 3.94 (br s, 1H, NHC(O)), 5.8 (d,
2
(d, 1H, JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.70 (d, 1H,
2
3JHH = 10.6 Hz, CHCH(CH3)2), 6.12 (d, 1H, JHH = 15.7 Hz, roofing
2
diastereotopic PhCH2), 7.20 (s, 4H, 4 ꢂ Ar-CH, tolyl group), 7.26
1H, JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.68 (d, 1H,
2
3JHH = 10.5 Hz, CHCH(CH3)2), 6.12 (d, 1H, JHH = 15.7 Hz, roofing
(m, 2H, o-Ph overlapping with CHCl3 residual solvent resonance),
3
7.31 (m, 1H, p-Ph), 7.40 (m, 2H, m-Ph), 7.49 (dd, 1H, JHH = 8.6 Hz
diastereotopic PhCH2), 6.97–7.42 (m, 9H, Ar), 7.49 (dd, 1H,
4
4
3JHH = 8.6 Hz and w-coupling JHH = 1.8 Hz, Ar: –CH–CH–C(Cl)CH–
and w-coupling JHH = 2.0 Hz, Ar: -CH-CH-C(Cl)CH-), 7.76 (d, 1H,
4
4
w-coupling JHH = 2.0 Hz, Ar: CH–C(Cl)–CHC(N)), 8.32 (d, 1H,
), 7.76 (d, 1H, w-coupling JHH = 1.8 Hz, Ar: CH–C(Cl)–CHC(N)),
3JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)). 13C{1H} (100 MHz, CDCl3): dC
18.4 (CH3), 19.3 (CH3), 21.5 (PhCH3), 28.5 (3 ꢂ CH3), 29.0
3
8.32 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)). 13C{1H} (100 MHz,
CDCl3): dC 18.4 (CH3), 19.2 (CH3), 28.5 (3 ꢂ CH3), 29.0
(CHCH(CH3)2), 31.2 (–NCH2CH2CH2–), 37.7 (–NCH2CH2CH2–), 42.2
(–NCH2CH2CH2–), 45.7 (PhCH2), 59.8 (CHCH(CH3)2), 79.3 (OC(CH3)3
(CHCH(CH3)2:
HSQC),
30.9
(–NCH2CH2CH2–),
37.8
(–NCH2CH2CH2–), 42.1 (–NCH2CH2CH2–), 45.7 (PhCH2), 59.7
(CHCH(CH3)2), 79.4 (OC(CH3)3), 119.5 (IV° C), 126.1 (2C, Ar:
2 ꢂ CH3CH), 127.0 (Ar: CH–C(Cl)–CHC(N)), 127.5 (2 ꢂ o-Ph), 127.8
3
[weak]), 113.5 and 113.7 (1C, JHF = 23.0 Hz, Ar), 116.7 and 116.9
3
(1C, JHF = 20.8 Hz, Ar), 119.5 (IV° C), 121.8 (Ar), 127.0 (Ar:

504
J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
CH–C(Cl)–CHC(N)), 127.3 (2 ꢂ o-Ph), 127.9 (p-Ph), 128.4 (Ar: –CH–
CH–C(Cl)CH–), 128.9 (2 ꢂ m-Ph), 129.1 (Ar: –CH–CH–C(Cl)CH),
(CHCH(CH3)2), 79.8 (OC(CH3)3), 119.5 (IV° C), 124.2 (2 ꢂ CH–
ArNO2), 127.0 (Ar: CH–C(Cl)–CHC(N)), 127.2 (2 ꢂ o-Ph), 127.3
(2 ꢂ CH–ArNO2), 128.0 (p-Ph), 128.5 (Ar: –CH–CH–C(Cl)CH–),
129.0 (2 ꢂ m-Ph), 129.1 (Ar: –CH–CH–C(Cl)CH), 136.7 (IV° C),
141.1 (IV° C), 142.8 (IV° C), 147.6 (IV° C), 155.4 (IV° C), 162.2 (IV°
C), 170.9 (IV° C). MS m/z (MeCN) 648 (100%) [M+H+]. HRMS-ESI
3
130.7 and 130.8 (1C, JHF = 8.0 Hz, Ar), 136.8 (IV° C), 138.6 and
3
138.7 (1C, JHF = 6.9 Hz, IV° C), 141.1 (IV° C), 147.7 (IV° C), 155.8
(IV° C), 161.3 (IV° C), 162.2 (IV° C), 163.8 (IV° C), 171.5 (IV° C).
19F NMR (376 MHz, CDCl3; CFCl3 reference): dF ꢁ110.78 ppm. MS
m/z (MeCN) 621 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH)
found 621.2645, calcd for C34H39ClFN4O4 621.2638 [M+H+]. Analyt-
ical data for the synthesis of 16-(R) and 16-(S) were identical
m/z (DCM/MeOH) found 648.2582, calcd for
C34H39ClN5O6
648.2583 [M+H+]. Analytical data for the synthesis of 18-(R) and
18-(S) were identical.
4.1.4. ( )-tert-Butyl (3-(N-(1-(3-benzyl-7-chloro-4-oxo-3,4-
dihydroquinazolin-2-yl)-2-methylpropyl)-2-fluorobenzamido)
propyl)carbamate 17
4.1.6. General method B
Deprotection of BOC-protect derivatives of ispinesib was
accomplished using a standard trifluoroacetic acid method. Details
are presented for the synthesis of ( )-N-(3-aminopropyl)-N-(1-(3-
benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpro-
pyl)-4-methylbenzamide, racemate 19 (ispinesib).
Compound 13 (0.10 g, 0.20 mmol) was reacted with o-fluor-
obenzoyl chloride (0.044 g, 0.28 mmol, 1.4 equiv) in accordance
with general method A to give compound 17 (0.110 g, 0.177 mmol,
89%). 1H NMR (400 MHz, CDCl3): dH 0.38 (d, 3H, 3JHH = 6.3 Hz, mag-
netically inequivalent CH(CH3)2), 0.77 (m, 1H, –CH2CH2CH2 diaste-
Compound 14 (0.139 g, 0.226 mmol) was added to a flame dried
25 ml round bottom flask, placed under N2(g) atmosphere and dis-
solved in anhydrous dichloromethane (2 mL). Trifluoroacetic acid
(TFA; 2 mL) was added and the reaction was stirred at rt for 2 h.
The reaction was monitored by TLC (30% EtOAc/hexanes/ꢀ0.1%
conc. NH3, UV) with the product observed as the major spot at
the baseline Rf = 0.00 and the starting compound 14 at Rf = 0.50.
Solvents were then removed under reduced pressure and the crude
residue was dissolved in dichloromethane (20 mL), washed with
sat. NaHCO3(aq) (2 ꢂ 15 mL), brine (15 mL). The organic fraction
was separated, dried over Na2SO4(s) for 30 min, filtered and then
the solvent was removed under reduced pressure and the sample
dried in vacuo to give purified compound 19, ( )-ispinesib, as a
white solid (0.105 g, 0.20 mmol, 88%). 1H NMR (400 MHz, CDCl3):
3
reotopic), 0.96 (d, 3H, JHH = 6.8 Hz, magnetically inequivalent
CH(CH3)2), 1.41 (br s, 10H, overlapping 9H, –OC(CH3)3 and 1H, -
CH2CH2CH2 diastereotopic), 2.58–2.77 (m, 3H, overlapping reso-
nances: 1H, CH2CH(CH3)2, and 2H, NCH2CH2CH2–), 3.23–3.52 (m,
2H, NCH2CH2CH2–, diastereotopic), 3.95 (br s, 1H, NHC(O)), 5.13
2
(d, 1H, JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.70 (d, 1H,
2
3JHH = 10.6 Hz, CHCH(CH3)2), 6.16 (d, 1H, JHH = 15.7 Hz, roofing
diastereotopic PhCH2), 7.12–7.42 (m, 9H, Ar), 7.49 (dd, 1H,
4
3JHH = 8.6 Hz and w-coupling JHH = 2.0 Hz, Ar: –CH–CH–C(Cl)CH–
4
), 7.77 (d, 1H, w-coupling JHH = 2.0 Hz, Ar: CH–C(Cl)–CHC(N)),
3
8.33 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)). 13C{1H} (100 MHz,
CDCl3): dC 18.2 (CH3), 19.2 (CH3), 28.4 (3 ꢂ CH3), 28.8
(CHCH(CH3)2), 30.7 (–NCH2CH2CH2–), 37.7 (–NCH2CH2CH2–), 41.8
(-NCH2CH2CH2-), 45.6 (PhCH2), 59.8 (CHCH(CH3)2), 79.4 (OC(CH3)3
3
dH 0.38 (d, 3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2),
0.81–0.88 (m, 1H, –CH2CH2CH2 diastereotopic), 0.96 (d, 3H,
3JHH = 6.7 Hz, magnetically inequivalent CH(CH3)2), 1.32–1.33 (m,
1H, –CH2CH2CH2 diastereotopic), 2.17–2.21 (m, 2H,
NCH2CH2CH2–), 2.36 (s, 3H, PhCH3), 2.70–2.79 (m,
1H, CH2CH(CH3)2), 3.35–3.50 (m, 2H, NCH2CH2CH2–), 5.22 (d, 1H,
2JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.71 (d, 1H,
3
[weak]), 116.2 and 116.4 (1C, JHF = 20.9 Hz, Ar), 119.4 (IV° C),
3
124.69 and 124.72 (1C, JHF = 3.1 Hz, Ar), 124.8 and 125.0 (1C,
3JHF = 18.4 Hz, Ar), 126.9 (Ar: CH–C(Cl)–CHC(N)), 127.3 (2 ꢂ o-Ph),
127.8 (p-Ph), 128.3 (Ar: –CH–CH–C(Cl)CH–), 128.8 (2 ꢂ m-Ph),
3
129.0 (Ar: –CH–CH–C(Cl)CH), 131.3 and 131.4 (1C, JHF = 7.7 Hz,
2
Ar), 136.8 (IV° C), 140.9 (IV° C), 147.6 (IV° C), 155.7 (IV° C), 156.4
(IV° C), 158.9 (IV° C), 162.2 (IV° C), 168.4 (IV° C), 171.2 (IV° C).
19F NMR (376 MHz, CDCl3; CFCl3 reference): dF ꢁ114.22 ppm. MS
m/z (MeCN) 621 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH)
found 621.2644, calcd for C34H39ClFN4O4 621.2638 [M+H+]. Analyt-
ical data for the synthesis of 17-(R) and 17-(S) were identical.
3JHH = 10.6 Hz, CHCH(CH3)2), 6.11 (d, 1H, JHH = 15.7 Hz, roofing
diastereotopic PhCH2), 7.18–7.23 (m, 4H, 4 ꢂ Ar-CH, tolyl group),
7.27 (m, 2H, o-Ph overlapping with CHCl3 residual solvent reso-
nance), 7.31 (m, 1H, p-Ph), 7.40 (m, 2H, m-Ph), 7.46 (dd, 1H,
3JHH = 8.6 Hz and w-coupling JHH = 2.0 Hz, Ar: –CH–CH–C(Cl)CH–
4
4
), 7.69 (d, 1H, w-coupling JHH = 2.0 Hz, Ar: CH–C(Cl)–CHC(N)),
8.30 (d, 1H, 3JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)) NB: NH2 proton res-
onance was not observed. 13C{1H} (100 MHz, CDCl3): dC 18.5 (CH3),
19.3 (CH3), 21.5 (PhCH3), 29.0 (CHCH(CH3)2: HSQC), 33.4
(–NCH2CH2CH2–), 39.3 (–NCH2CH2CH2–), 42.3 (–NCH2CH2CH2–),
45.7 (PhCH2), 59.7 (CHCH(CH3)2), 119.5 (IV° C), 126.2 (2C, Ar:
2 ꢂ CH3CHCH), 127.0 (Ar: CH–C(Cl)–CHC(N)), 127.4 (2 ꢂ o-Ph),
127.8 (p-Ph), 128.2 (Ar: –CH–CH–C(Cl)CH–), 128.9 (2C, Ar:
2 ꢂ CH3CHCH: HMBC), 129.1 (Ar: –CH–CH–C(Cl)CH), 129.4
(2 ꢂ m-Ph), 134.0 (IV° C), 137.0 (IV° C), 139.7 (IV° C), 140.9 (IV°
C), 147.8 (IV° C), 156.1 (IV° C), 162.4 (IV° C), 173.2 (IV° C). MS m/
z (MeCN) 517 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH) found
4.1.5. (R)-tert-Butyl (3-(N-(1-(3-benzyl-7-chloro-4-oxo-3,4-
dihydroquinazolin-2-yl)-2-methylpropyl)-4-nitrobenzamido)
propyl)carbamate 18
Compound 13-(R) (0.150 g, 0.30 mmol) was reacted with p-
nitrobenzoyl chloride (0.078 g, 0.42 mmol, 1.4 equiv) in accor-
dance with general method A to give compound 18 (0.176 g,
0.272 mmol, 91%). 1H NMR (400 MHz, CDCl3): dH 0.39 (d, 3H,
3JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2), 0.80 (m, 2H, –
3
CH2CH2CH2), 0.85 (d, 3H, JHH = 7.3 Hz, magnetically inequivalent
CH(CH3)2), 1.38 (s, 9H, –OC(CH3)3), 2.55–2.81 (m, 3H, overlapping
resonances: 1H, CH2CH(CH3)2, and 2H, NCH2CH2CH2–), 3.22–3.54
(m, 2H, NCH2CH2CH2– diastereotopic), 4.00 (br s, 1H, NHC(O)),
517.2359, calcd for C30H34ClN4O42 517.2365 [M+H+]. [
a] (25 °C;
D
CHCl3; 19-(R)) = +345.9 0.4. Analytical data for the synthesis of
19-(R) and 19-(S) were identical.
2
5.17 (d, 1H, JHH = 15.8 Hz, roofing diastereotopic PhCH2), 5.71 (d,
3
2
1H, JHH = 10.6 Hz, CHCH(CH3)2), 6.13 (d, 1H, JHH = 15.8 Hz,
roofing diastereotopic PhCH2), 7.28-7.45 (m, 7H, 2 ꢂ Ar-CH, o-Ph,
4.1.7. ( )-N-(3-aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-
3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-4-
fluorobenzamide 20
3
m-Ph and p-Ph), 7.50 (dd, 1H, JHH = 8.6 Hz and w-coupling
4JHH = 2.2 Hz, Ar: –CH–CH–C(Cl)CH–), 7.77 (d, 1H, w-coupling
4JHH = 2.2 Hz, Ar: CH–C(Cl)–CHC(N)), 8.28 (d, 2H, 3JHH
=
Compound 15 (0.115 g, 0.185 mmol) was deprotected in accor-
dance with general method B to give compound 20 as a white solid
(0.078 g, 0.15 mmol, 81%). 1H NMR (400 MHz, CDCl3): dH 0.37 (d,
3
8.6 Hz, Ar), 8.33 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)).
13C{1H} (100 MHz, CDCl3): dC 18.5 (CH3), 19.2 (CH3), 28.4
(3 ꢂ CH3), 29.1 (CHCH(CH3)2: HSQC), 31.4 (–NCH2CH2CH2–), 37.6
(–NCH2CH2CH2–), 42.2 (–NCH2CH2CH2–), 45.8 (PhCH2), 59.8
3
3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2), 0.86 (m,
3
1H, –CH2CH2CH2 diastereotopic), 0.95 (d, 3H, JHH = 6.7 Hz,

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
505
magnetically inequivalent CH(CH3)2), 1.32 (m, 1H, –CH2CH2CH2
diastereotopic), 2.22 (m, 2H, NCH2CH2CH2–), 2.74 (m, 1H,
CH2CH(CH3)2), 3.33-3.53 (m, 2H, NCH2CH2CH2–), 5.18 (d, 1H,
2JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.70 (d, 1H,
(DCM/MeOH) found 521.2115, calcd for C29H31ClFN4O2 521.2114
[M+H+]. [
(25 °C; CHCl3; 22-(S)) = –355.6 0.4. Analytical data
for the synthesis of 22-(R) and 22-(S) were identical.
a]
D
2
3JHH = 10.5 Hz, CHCH(CH3)2), 6.11 (d, 1H, JHH = 15.7 Hz, roofing
4.1.10. Mosher’s amide synthesis
diastereotopic PhCH2), 7.07–7.39 (m, 9H, 4 ꢂ Ar-CH, o-Ph, m-Ph
The absolute configuration of the stereo-center in chirally re-
solved compound 10-(R) was assigned by synthesising the
Mosher’s amide derivatives 23-(R,R) and 23-(R,S) in accordance
with the methods described by Hoye et al.18,22
and p-Ph), 7.47 (dd, 1H, 3JHH = 8.6 Hz and w-coupling 4JHH = 2.0 Hz,
4
Ar: –CH–CH–C(Cl)CH–), 7.69 (d, 1H, w-coupling JHH = 2.0 Hz, Ar:
3
CH–C(Cl)–CHC(N)), 8.29 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–
C(Cl)). 13C{1H} (100 MHz, CDCl3): dC 18.5 (CH3), 19.3 (CH3), 29.1
(CHCH(CH3)2:
HSQC),
32.6
(–NCH2CH2CH2–),
39.0
(–
4.1.11. Synthesis of compound 23-(R,R)
NCH2CH2CH2NH2), 42.3 (–NCH2CH2CH2NH2), 45.7 (PhCH2), 59.9
(CHCH(CH3)2), 115.9 and 116.1 (2C, Ar: 2 ꢂ F-CCH), 119.5 (IV° C),
127.0 (Ar: CH–C(Cl)–CHC(N)), 127.3 (2 ꢂ o-Ph), 127.9 (p-Ph),
128.3 and 128.35 (2C, Ar: 2 ꢂ F-CCHCH), 128.4 (Ar: –CH–CH–
C(Cl)CH–), 128.9 (2 ꢂ m-Ph), 129.1 (Ar: –CH–CH–C(Cl)CH), 132.9
(IV° C), 136.9 (IV° C), 141.0 (IV° C), 147.7 (IV° C), 155.8 (IV° C),
162.3 (IV° C), 164.4 (IV° C), 172.1 (IV° C). 19F NMR (376 MHz,
CDCl3; CFCl3 reference): dF ꢁ110.28 ppm. MS m/z (MeCN) 521
(100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH) found 521.2105, calcd
NB: The absolute configuration of the stereo-center in resolved
compound 10-(R) was not known prior to Mosher’s amide analysis.
Compound numbers are given here for clarity and reflect the re-
sults of this analysis. To a 10 mL reaction vial was added compound
10-(R) (0.025 g, 7.3 ꢂ 10ꢁ5 mol), (R)-(+)-
a-methoxy-a-trifluoro-
methylphenyl acetic acid (R-(+)-MTPA-OH; 0.0553 g, 2.36 ꢂ
10ꢁ4 mol, 3.23 equiv), anhydrous pyridine (.023 g, 2.9 ꢂ 10ꢁ4 mol,
4 equiv) in anhydrous dichloromethane (total of 1.0 mL). Then
N,N0-dicyclohexylcarbodiimide (DCC; 0.060 g, 2.9 ꢂ 10ꢁ4 mol,
4 equiv) was added at rt with stirring upon which a white precip-
itate formed immediately. The reaction was left to stir at rt for 24 h
and monitored by TLC (20% EtOAc/hexanes, UV) with the product
observed as the major spot at Rf = 0.40. Then the reaction was fil-
tered through a cotton plug to remove the insoluble dicyclohexyl-
urea (DCU) by-product before partitioning the filtrate between
water (5 mL) and diethyl ether (10 mL). The mixture was separated
and the aqueous fraction was extracted with diethyl ether
(3 ꢂ 10 mL). The organic fractions were combined, dried over anhy-
drous Na2SO4(s) for 30 min, filtered and then the solvent was re-
moved under reduced pressure. The crude mixture was purified
on a small silica gel column (15% EtOAc/hexane), fractions contain-
ing product were identified by TLC, pool and dried the solvent was
removed under reduced pressure. The final compound was dried
in vacuo to give compound 23-(R,R) as a clear light pink coloured
oil (0.0318 g, 5.7 ꢂ 10ꢁ5 mol, 78%). NB: Only selected NMR data used
in the analysis of the absolute configuration are presented. Resonance
assignments correspond with those listed in Scheme 2. 1H NMR
for C29H31ClFN4O2 521.2114 [M+H+].
[a
]
D
(25 °C; CHCl3; 20-
(R)) = +306.4 0.8. Analytical data for the synthesis of 20-(R) and
20-(S) were identical.
4.1.8. ( )-N-(3-Aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-
3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-3-
fluorobenzamide 21
Compound 16 (0.079 g, 0.127 mmol) was deprotected in accor-
dance with general method B to give compound 21 as a white solid
(0.053 g, 0.102 mmol, 80%). 1H NMR (400 MHz, CDCl3): dH 0.39 (d,
3
3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2), 0.85 (m,
3
1H, –CH2CH2CH2 diastereotopic), 0.95 (d, 3H, JHH = 6.8 Hz, mag-
netically inequivalent CH(CH3)2), 1.30 (m, 1H, -CH2CH2CH2 diaste-
reotopic), 2.22 (m, 2H, NCH2CH2CH2–), 2.76 (m, 1H, CH2CH(CH3)2),
2
3.43 (m, 2H, NCH2CH2CH2–), 5.17 (d, 1H, JHH = 15.6 Hz, roofing
3
diastereotopic PhCH2), 5.70 (d, 1H, JHH = 10.6 Hz, CHCH(CH3)2),
2
6.13 (d, 1H, JHH = 15.6 Hz, roofing diastereotopic PhCH2), 7.02-
7.40 (m, 9H, 4 ꢂ Ar-CH, o-Ph, m-Ph and p-Ph), 7.48 (dd, 1H,
4
3
3JHH = 8.6 Hz and w-coupling JHH = 2.0 Hz, Ar: –CH–CH–C(Cl)CH–
(400 MHz, CDCl3): dH 0.385 (d, 3H, JHH = 6.7 Hz, magnetically
4
), 7.69 (d, 1H, w-coupling JHH = 2.0 Hz, Ar: CH–C(Cl)–CHC(N)),
inequivalent CH(CH3)2 [a]), 0.736 (d, 3H, 3JHH = 6.7 Hz, magnetically
3
8.30 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–C(Cl)). 19F NMR
inequivalent CH(CH3)2 [a⁄]), 2.09 (m, 1H, CH2CH(CH3)2 [b]), 5.16 (m,
2
(376 MHz, CDCl3; CFCl3 reference): dF -111.3 ppm. MS m/z (MeCN)
1H, CHCH(CH3)2 [c]), 5.28 (d, 1H, JHH = 15.7 Hz, roofing diastereo-
521 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH) found 521.2099,
topic PhCH2 [g]), 5.81 (d, 1H, JHH = 15.7 Hz, roofing diastereotopic
2
calcd for C29H31ClFN4O2 521.2114 [M+H+]. [
a
]
D
(25 °C; CHCl3; 21-
PhCH2 [g⁄]), 8.28 (d, 1H, JHH = 8.6 Hz, Ar: C-CH-CH-C(Cl) [f]). MS
3
(S)) = ꢁ339.7 0.8. Analytical data for the synthesis of 21-(R) and
m/z (MeCN) 558 (100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH) found
21-(S) were identical.
558.1771, calcd for C29H28ClF3N3O3 558.1766 [M+H+].
4.1.9. ( )-N-(3-Aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-
3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-2-
fluorobenzamide 22
4.1.12. Synthesis of compound 23-(R,S)
With the exception of using (S)-(ꢁ)-
a-methoxy-a-trifluoro-
methylphenyl acetic acid, S-(ꢁ)-MTPA-OH, the same procedure gi-
ven for the synthesis of compound 23-(R,R) was used to give
compound 23-(R,S) as a clear light pink coloured oil (0.041 g,
7.35 ꢂ 10ꢁ5 mol, 100%). 1H NMR (400 MHz, CDCl3): dH 0.44 (d,
Compound 17 (0.107 g, 0.172 mmol) was deprotected in accor-
dance with general method B to give compound 22 as a white so-
lid(0.073 g, 0.140 mmol, 81%). 1H NMR (400 MHz, CDCl3): dH 0.39
3
3
(d, 3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2), 0.83
3H, JHH = 6.2 Hz, magnetically inequivalent CH(CH3)2 [a]), 0.91
3
3
(m, 1H, –CH2CH2CH2 diastereotopic), 0.96 (d, 3H, JHH = 6.8 Hz,
(d, 3H, JHH = 6.4 Hz, magnetically inequivalent CH(CH3)2 [a⁄]),
magnetically inequivalent CH(CH3)2), 1.26 (m, 1H, –CH2CH2CH2
diastereotopic), 2.18 (m, 2H, NCH2CH2CH2–), 2.75 (m, 1H,
CH2CH(CH3)2), 3.38 (m, 2H, NCH2CH2CH2–), 5.11 (d, 1H,
2JHH = 15.7 Hz, roofing diastereotopic PhCH2), 5.70 (d, 1H,
2.14 (m, 1H, CH2CH(CH3)2 [b]), 5.13 (m, 1H, CHCH(CH3)2 [c]),
2
5.29 (d, 1H, JHH = 15.6 Hz, roofing diastereotopic PhCH2 [g]), 5.77
2
(d, 1H, JHH = 15.6 Hz, roofing diastereotopic PhCH2 [g⁄]), 8.27 (d,
3
1H, JHH = 8.6 Hz, Ar: C-CH-CH-C(Cl) [f]). MS m/z (MeCN) 558
3JHH = 10.6 Hz, CHCH(CH3)2), 6.13 (d, 1H, JHH = 15.7 Hz, roofing
(100%) [M+H+]. HRMS-ESI m/z (DCM/MeOH) found 558.1772, calcd
2
diastereotopic PhCH2), 7.10–7.41 (m, 9H, 4 ꢂ Ar-CH, o-Ph, m-Ph
for C29H28ClF3N3O3 558.1766 [M+H+].
and p-Ph), 7.48 (dd, 1H, 3JHH = 8.6 Hz and w-coupling 4JHH = 2.0 Hz,
4
Ar: –CH–CH–C(Cl)CH–), 7.69 (d, 1H, w-coupling JHH = 2.0 Hz, Ar:
4.2. Single-crystal X-ray diffraction
3
CH–C(Cl)–CHC(N)), 8.31 (d, 1H, JHH = 8.6 Hz, Ar: C–CH–CH–
C(Cl)). 19F NMR (376 MHz, CDCl3; CFCl3 reference): dF
ꢁ114.71 ppm. MS m/z (MeCN) 521 (100%) [M+H+]. HRMS-ESI m/z
Single crystal X-ray diffraction data were obtained for com-
pounds 3, 7 and 9. In each case, a typical crystal was mounted

506
J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
using the oil drop technique, in perfluoropolyether oil at 150(2) K
using a Cryostream N2 open-flow cooling device.23 Diffraction data
the exception of MCF-7, where cell density was 1000 cells/well,
and allowed to adhere for 24 h. Cells were then exposed to the
compounds which were added directly to the media. Appropriate
controls for culture media and vehicle were used throughout to
measure non-specific background, and to serve as a reference to
each cell line. Plates were incubated at 37 °C for 44 h then treated
with MTT solution and cultured for 3 h. After removing the media,
were collected using graphite monochromatic Mo-K radiation
a
(k = 0.71073 Å) on a Nonius Kappa CCD diffractometer. For all data
collections, series of
x-scans were performed in such a way as to
collect a complete data set to a maximum resolution of 0.77 Å. Data
reduction including unit cell refinement and inter-frame scaling
was carried out using DENZO-SMN/SCALEPACK.24 Intensity data
were processed and corrected for absorption effects by the multi-
scan method, based on repeat measurements of identical and Laue
equivalent reflections. Structure solution was carried out with di-
rect methods using the program SIR9225 within the CRYSTALS soft-
ware suite.26 In general, coordinates and anisotropic displacement
parameters of all non-hydrogen atoms were refined freely except
where disorder necessitated the use of ‘‘same distance restraints’’
together with thermal similarity and vibrational restraints to
maintain sensible geometry/displacement parameters. Hydrogen
atoms were generally visible in the difference map and refined
with soft restraints prior to inclusion in the final refinement using
a riding model.27 Crystallographic data (excluding structure fac-
tors) for all the structures have been deposited with the Cambridge
Crystallographic Data Centre (CCDC: 897765–897767). Copies of
these data can be obtained free of charge from The Cambridge
Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re-
quest/cif. A summary of the X-ray crystallographic data is provided
in Supplementary data Table S1.
DMSO (100 lL/well) was added to solubilise the purple formazan
product. Absorbance was then measured at 560 nm by using Perkin
Elmer Victor™ X3 Multilabel Plate Reader. Dose–response curves
following either a monophasic or biphasic profile were determined
with GraphPad Prism 5.01 (GraphPad Software, La Jolla, CA, USA)
by non-linear regression analysis of
(560 nm) versus drug concentration.
a plot of absorbance
BrdU assays were conducted in a similar manner to the MTT as-
says and were analysed by using the Cell Proliferation ELISA BrdU
assay kit (Roche Applied Science, Mannheim, Germany) in accor-
dance with the manufacturer’s protocol.
4.6. Flow cytometry
Cells were plated onto 100 mm2 dishes at a cell density of
1.0 ꢂ 106 cells/dish and 24 h later, were treated with 100 nM of
the compounds. 24 h after treatment, cells were washed once with
ice-cold PBS (5 mL), detached by treatment with trypsin/EDTA
solution and centrifuged for 5 min at 1000 rpm at rt. The cell pellet
was washed twice with 10 mL PBS, resuspended in 4 mL of ice-cold
fixation solution (70 vol% ethanol, 30 vol% H2O) and stored at
ꢁ20 °C for 24 h. Thereafter, cold PBS (10 mL) was added to the cell
suspension before centrifugation for 5 min at 1000 rpm. The cell
pellet was washed once with PBS (10 mL; 4 °C) and resuspended
in 1.0 mL of staining solution containing 0.1 mg/mL RNAse A (Sig-
4.3. Kinesin motor protein ATP-hydrolase inhibition assay
Inhibition of the ATP-hydrolase activity of various kinesin mo-
tor proteins was measured by using the Kinesin ATPase End-Point
Biochem Kit, BK053 (Cytoskeleton, Denver, USA). All motor pro-
teins were also obtained from Cytoskeleton. Generation of inor-
ganic phosphate (Pi) during MT-activated ATP-hydrolase activity
of kinesin motor proteins was measured at 650 nm. The concentra-
tion of the motor proteins (2.5 lg total protein) used in the assay
was kept as low as possible to minimise possible crowding effects
on MTs. Compounds were diluted to give final concentrations of
1.0 lM and were incubated at rt for 10 to 20 min. Assays were con-
ma Aldrich), 50 lg/mL propidium iodide (PI) from 2.5 mg/mL stock
solution and 0.05% Triton X-100 (Fluka) in PBS. After incubation at
37 °C for 40 min in the dark, cells were washed in PBS, and ana-
lysed on a guava easyCyte HT2L flow cytometry system (Millipore).
Data were analysed by cell-cycle analysis software (Flowjo, Tree-
Star Inc.). For phospho-histone H3 analysis, fixed cells were incu-
bated with a mouse anti-phospho-histone H3 (Ser10) antibody
(1:50 dilution in PBS, Cell Signalling Technology, BioConcept,
Allschwil, Switzerland) for 45 min at rt. The cells were washed
twice with PBS and incubated with a FITC-conjugated goat anti-
mouse IgG (1:200 dilution in PBS, Sigma–Aldrich, Buchs,
Switzerland) for 30 min in the dark. Cells were stained with PI as
described above before flow cytometric analysis.
ducted in accordance with the manufacturer’s protocol.
4.4. Cells and cell culture
Cell culture media and additives were obtained from BioCon-
cept (Allschwil, Switzerland). PC-3 human prostate adenocarci-
noma and SKOV3 human ovary adenocarcinoma cells were
obtained from the European Collection of Cell Cultures (ECACC,
Salisbury, UK) and were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) with 10% Fetal Calf Serum (FCS) (v/v). DU-145
human prostate carcinoma cells were cultured in Minimum Essen-
tial Medium Eagle (MEM) containing sodium pyruvate (1 mM), so-
dium bicarbonate (1.5 g/L) and 1% non-essential amino acids
(NEAA). MCF-7 human breast cancer epithelial cells were cultured
in a 1:1 mixture of DMEM/Ham’s F-12 medium. All cell culture
media were supplemented with 10% (v/v) fetal calf serum (FCS),
glutamine (2 mM), and antibiotics (penicillin [100 units/mL],
4.7. Confocal fluorescence microscopy
4.7.1. Permeabilisation buffer
Permeabilisation buffer was freshly prepared from a 10ꢂ con-
centrated stock solution (1.54 M NaCl, 15.44 mM KH2PO4,
28.58 mM Na2HPO4ꢃ7H2O and 5% Triton X-100). To 10ꢂ permeabil-
isation buffer (5 mL) was added deionised H2O (40 mL). The pH
was adjusted to pH 7.2, and then the solution was diluted to
50 mL to make a 1.0ꢂ permeabilisation buffer stock solution. Stock
solutions can be kept for several weeks in a refrigerator. To 1.0ꢂ
permeabilisation buffer (2.4 mL) was added PBS (9.6 mL) to give
0.2ꢂ permeabilisation buffer used as working solution.
streptomycin [100 lg/mL] and fungizone [0.25 lg/mL]). All cell
lines were maintained at 37 °C with 5% carbon dioxide.
4.5. In vitro cell proliferation assays
4.7.2. Blocking buffer
A solution of PBS containing 1% BSA and 0.3% Tween-20 was
prepared.
Compounds were dissolved in DMSO in concentrations ranging
from 1.0 to 1.0 mM. The compounds were serially diluted (typically
1:4 or 1:2 dilution) with growth medium then diluted into the cell
assay plate with a final DMSO concentration of 60.2%. Cells were
plated in 96-well plates with a density of 2000 cells/well with
4.7.3. Confocal fluorescence microscopy
Cells were maintained at 37 °C with 5% carbon dioxide and were
plated in 8-well Lab-Tek II Chamber Slides (NUNC, VWR, LabShop,

J. P. Holland et al. / Bioorg. Med. Chem. 21 (2013) 496–507
507
Batavia, IL) with a density of 5000 cells/well and allowed to adhere
for 24 h and to grow to 80% confluence. Cells were then exposed to
0.1 M concentrations of compounds 20–22 and incubated for 22 h
at 37 °C. All plates contained wells with culture media only to
serve as a reference control to the SKOV-3 cell line.
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a
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l
a
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Acknowledgments
Funded in part by the ETH Fellowship Program (FEL-01-10-1;
JPH). We thank Dr. NagaVaraKishore Pillarsetty, Dr. Aristeidis Chio-
tellis, Dr. Selena Melicevic Sephton and Cindy Fischer for helpful
discussions and advice regarding chemical synthesis and chroma-
tography. We also thank Dr. Tobias Schwarz and Dr. Joaquim Hehl
at the Light Microscopy Centre, ETH Zurich for technical support
regarding fluorescence microscopy studies.
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.013.